Factor IX p.A37V mutation causes severe bleeding in a patient with phenprocoumon therapy

Background Bleeding is the most common complication of oral anticoagulants, due to inadequate dosing. Case presentation This report describes the clinical course of a patient who developed severe bleeding under therapy with phenprocoumon, despite an INR in the lower therapeutic range. Strikingly, aPTT was prolonged, while factor IX activity was significantly reduced. Acquired hemophilia was excluded, due to missing detection of inhibitors. Finally, sequencing part of the factor IX gene including nucleotide position c.110 revealed a hemizygous factor IX mutation c.110C > T p (Ala37Val). Conclusions In rare cases, missense mutations in factor IX propeptide are associated with severe bleeding complications. The substitution of alanin at position 37 to either valin or threonin (Ala37Val or Ala37Thr) leads to hypersensitivity to vitamin k antagonists.

A rapid, cost efficient and simple method to identify current SARS-CoV-2 variants of concern by Sanger sequencing part of the spike protein gene

In 2020, the novel coronavirus, SARS-CoV-2, caused a pandemic, which is still raging at the time of writing this. Many countries have set up high throughput RT-qPCR based diagnostics for people with COVID-19 symptoms and for the wider population. In addition, with the use of whole genome sequencing (WGS) new lineages of SARS-CoV-2 have been identified that have been associated with increased transmissibility or altered vaccine efficacy, so-called Variants of Concern (VoC). WGS is generally too labor intensive and expensive to be applied to all positive samples from the diagnostic tests, and often has a turnaround time too long to enable VoC focused contact tracing. Here, we propose to use Sanger sequencing for the detection of common variants of concern and key mutations in early 2021, using a single set of the recognized ARTIC Network primers. The proposed setup relies entirely on materials and methods already in use in diagnostic RT-qPCR labs and on existing infrastructure from companies that have specialized in cheap and rapid turnaround Sanger sequencing. In addition, we provide an automated mutation calling software (https://github.com/kblin/covid-spike-classification). We have validated the setup on 195 SARS-CoV-2 positive samples, and we were able to profile >85% of RT-qPCR positive samples, where the last 15% largely stem from samples with low viral count. At approximately 4 € per sample in material cost, with minimal hands-on time, little data handling, and a turnaround time of less than 30 hours, the setup is simple enough to be implemented in any SARS-CoV-2 RT-qPCR diagnostic lab. Our protocol provides results that can be used to focus contact-tracing efforts and it is cheap enough for the tracking and surveillance of all positive samples for emerging variants such as B.1.1.7, B.1.351 and P.1 as of January 2021.

Identification of Pathogenic Genes of Nonsyndromic Hearing Loss in Uyghur Families Using Massively Parallel DNA Sequencing Technique

We aim to identify the mutations of deafness genes using massively parallel DNA sequencing in the 12 Uyghur families. SNPscan method was used to screen against the 124 sites in the common deafness genes in probands. Subjects with SNPscan negativity were subject to massively parallel DNA sequencing for the sequencing of 97 genes known to be responsible for hearing loss. Eight families (66.7%) showed biallelic mutations in probands, including MYO15A mutation (6892C>T in J02 family, 9514C>T/7894G>T in J07 family, and 9514C>T in J16 family), MYO7A mutation (1258A>T in J03 family), TMC1 mutation (773G>A in J09 family and 1247T>G/1312G>A in J11 family), and PCDH15 mutation (4658delT in J08 and J13 families). Six novel types of mutation were identified including 6892C>T, 9514C>T/7894G>T, and 9514C>T in MYO15A gene, 1258A>T in MYO7A, 773G>A in TMC1, and 4658delT in PCDH15. The ratio of nonsense mutation and frameshift mutation was comparatively high. All these indicated that the mutation types reported in this study were rare. In conclusion, rare deafness genes were identified in the Uyghur families using massively parallel DNA sequencing, part of which were suggested to be related to the pathogenesis of the disease.

New Insight Into the Biology, Risk Stratification, and Targeted Treatment of Myelodysplastic Syndromes

In myelodysplastic syndromes (MDS), somatic mutations occur in five major categories: RNA splicing, DNA methylation, activated cell signaling, myeloid transcription factors, and chromatin modifiers. Although many MDS cases harbor more than one somatic mutation, in general, there is mutual exclusivity of mutated genes within a class. In addition to the prognostic significance of individual somatic mutations, more somatic mutations in MDS have been associated with poor prognosis. Prognostic assessment remains a critical component of the personalization of care for patient with MDS because treatment is highly risk adapted. Multiple methods for risk stratification are available with the revised International Prognostic Scoring System (IPSS-R), currently considered the gold standard. Increasing access to myeloid gene panels and greater evidence for the diagnostic and predictive value of somatic mutations will soon make sequencing part of the standard evaluation of patients with MDS. In the absence of formal guidelines for their prognostic use, well-validated mutations can still refine estimates of risk made with the IPSS-R. Not only are somatic gene mutations advantageous in understanding the biology of MDS and prognosis, they also offer potential as biomarkers and targets for the treatment of patients with MDS. Examples include deletion 5q, spliceosome complex gene mutations, and TP53 mutations.

Skin swabbing of amphibian larvae yields sufficient DNA for efficient sequencing and reliable microsatellite genotyping

Skin swabbing, a minimally invasive DNA sampling method recently developed on adult amphibians, was tested on larvae of fire salamanders (Salamandra salamandra). The quality and quantity of the sampled DNA was evaluated by (i) measuring DNA concentration in DNA extracts, (ii) sequencing part of the mtDNA cytochrome b gene (692 bp) and (iii) genotyping eight polymorphic nuclear microsatellite loci. The multiple-tubes approach was used for calculating allelic dropout (ADO) and false allele (FA) rates to evaluate the reliability of the genotypes. DNA extracts from tissue samples of road-killed individuals were included in the study as positive controls. Our results showed that skin swabs of fire salamander larvae can provide DNA in sufficient quantity and quality, as sequencing was successful and no allelic dropouts or false alleles were detected. This method, tested for the first time on amphibian larvae, has proven to be an efficient and reliable alternative to the controversial tail fin clipping procedure.

Spectrum of causative mutations in patients with haemophilia A in Austria

SummaryIn patients with haemophilia A knowledge of the pathogenetic mutation is important i) as basis for carrier diagnosis and ii) for risk estimation of inhibitor formation. The pathogenetic mutations were identified by testing inversions in intron 1 and 22 (IVS22 and IVS1) and sequencing part of the promoter, the coding region and the exon/intron boundaries in a cohort of Austrian haemophilia A patients. A total of 239 patients from nine participating centres, who had consented to genetic testing and of whom clinical information was available were included in the study. First, IVS22 and IVS1 were tested; in case of absence of either inversion patients were subjected to sequencing. Mutations within the FVIII gene were identified in 234 patients. Notably, 53 mutations had not previously been described in HAMSTeRS. Of our patient cohort, 72.5 % had either an IVS22 or a missense mutation. Interestingly, in three brothers with severe haemophilia, we found a double mutation in exon 14 (missense + small deletion). The spectrum of mutations in Austrian haemophilia A patients was comparable to that found in the German and Italian population; however, it differed from the spectrum reported in the UK. In conclusion, 53 not previously published mutations were identified in Austrian haemophilia A patients. The occurrence of double mutations in the factor VIII gene could be confirmed and their low frequency was corroborated. We speculate that the differences between mutations in Austria and other European countries are due to ethnic diversity. Detailed investigations of the association of ethnicity and the mutation spectrum are planned.